Among the number of different analytical methods that detect and quantify nucleic acid sequences, Polymerase Chain Reaction (PCR) has become the most powerful and wide-spread technology, the principles of which are disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,102 (Mullis et al.). However, a typical PCR reaction by itself only yields qualitative data, since, after a phase of exponential or progressive amplification, the amount of amplified nucleic acid reaches a plateau, such that the amount of generated reaction product is not proportional to the initial concentration of the template DNA.
As a consequence, many different PCR based protocols have been developed in order to obtain reliable and reproducible quantitative data. Generally, two different basic principles can be discriminated:
i) Competitive PCR using internal standards PA1 ii) Quantification of target DNA by initial generation of a calibration curve, reviewed by Siebert, in: Molecular Diagnosis of infectious diseases (ed. Reiscbl, Humana Press, Totowa, N.J., p. 55-79 (1998).
A major improvement in the generation of quantitative data derives from the possibility of measuring the kinetics of a PCR reaction by On-Line detection. This has become possible recently by means of detecting the amplicon through fluorescence monitoring. Examples of such techniques are disclosed in detail in WO 97/46707, WO 97/46712 and WO 97/46714 (Wittwer et al.), the disclosures of which are hereby incorporated by reference.
Prior to the availability of On-Line PCR-detection, Wiesner et al.(Nucl. Acids Res. 20, 5863-5864 (1992)), for the first time used data from multiple cycles of a PCR reaction, wherein after each cycle, the product concentration was assayed by radioactive incorporation and subsequent scintillation counting. For each curve, the initial template concentration (N.sub.o) and amplification efficiency (eff) were determined by linear regression of data points on a product concentration (Nn) versus cycle number graph as defined by the following formula: EQU Log Nn=(log eff)n+log N.sub.o.
Higuchi et al. (BioTechnology 11, 1026-2030 (1993) were the first to disclose a simpler approach for initial template quantification using fluorescence monitoring at each cycle. A fluorescence threshold level was used to define a fractional cycle number related to initial template concentration. Specifically, the log of the initial template concentration is inversely proportional to the fractional cycle number (CT), defined as the intersection of the fluorescence versus cycle number curve with the fluorescence threshold.
In a more sophisticated embodiment of the fluorescence threshold method, the threshold level is not chosen manually or arbitrarily, but is set to three times the coefficient of variation of the signals which are obtained as fluorescent background noise.
However, there exist several major draw backs of the referenced prior art. First of all, prior art methods require corrections for different fluorescent background signals in order to compensate for sample to sample variations due to differences in excitation or emission efficiencies. Secondly, since quantification of template dependent amplification always has to be performed during the log linear phase of the reaction (Kohler et al., "Quantitation of mRKA by PCR: Nonradioactive methods, p. 6 Springer Berlin 1995, ed. Kohler), a log linear window of the reaction has to be determined by the user prior to the actual quantification experiment. In addition, the setting of a threshold level either requires an experienced experimental person skilled in the art, since it is chosen manually without taking specific preconditions of the experiment to be performed into account.
Thus, there exists a requirement for methods, wherein the concentration of an amplifiable or replicable analyte may be determined without correction for different fluorescent background, independently from a user defined log phase and a user defined threshold level, and wherein at the same time the determined concentration is independent of the absolute level of signal which is generated in the plateau phase of the reaction. Moreover, such an independence of absolute signal level is also advantageous for systems, wherein multiple fluorescent signals being detected through multiple channels with different window ranges may be compared.